Characterization of protein-001 as a GTPase-activating protein for human ADP-ribosylation factor like-1 protein

• Main Authors: Tai Wei Lin, 林黛瑋
• Other Authors: C. J. Yu
• Format: Others
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/61718180413376226651

碩士 === 長庚大學 === 生物醫學研究所 === 101 === ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins are a distinct subfamily of Ras small GTPases. Like other Ras-related GTP-binding proteins, ARFs/ARLs are regulated by guanine-nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) to cycle between the active GTP-bound form in the membrane and the inactive GDP-bound form in the cytoplasm, respectively. ARL1 is enriched in the trans-Golgi network (TGN) and has been proposed to involve in intracellular vesicles trafficking. Neither GEFs nor GAPs have been identified for human ARL1. Previously, we used differential affinity purification combined with mass spectrometry to identify protein-001 as a potential GAP protein for human ARL1. Herein, we found that protein-001 was partially co-localized with ARL1 in TGN. Protein binding assay demonstrated that protein-001 directly interact with ARL1 in vitro. In vitro immunoprecipitation assay also demonstrated that the protein interaction between protein-001 and ARL1. Overexpression of wild-type, but not activity-deficiency protein-001 caused endogenous ARL1 dispersed from TGN specifically in HeLa cell. Furthermore, in vitro GAP activity assay revealed that protein-001 can hydrolysis GTP bound to ARL1. Collectively, our study showed the first time that the interaction and regulatory effects of protein-001 on human ARL1. Our results also suggest that protein-001 would be a novel GAP protein for human ARL1.